The treatment of cancer continues to be a problem for clinical and veterinary medicine. The treatment regimens available today include surgery, radiation, chemotherapy, immunotherapy (including autologous and heterologous cell therapy) or combinations thereof.
Surgery often fails due to tumor tissue being unrecognized and not removed. Radiation and chemotherapy also frequently fail, and the side effects of the treatments often decrease the quality of life for patients. Surgery and chemotherapy are associated with significant and often non-specific suppression of the immune system (Hammer et al., Eur. Surg. Res., 1992 24:133-137; Joos and Tam, Proc. Am. Thorac. Soc., 2005 2:445-448). This immune suppression is often associated with the occurrence of opportunistic infections, as exemplified by the known high rate of infectious complications in individuals undergoing high-dose whole-body irradiation (Gil et al., Infection, 2007 35:421-427). Radiation, surgery and chemotherapy are additionally associated with multilineage hematopoietic and myeloid suppression (myelosuppression) such as, but not limited, to leucopenia, neutropenia, thrombocytopenia and/or anemia (Montoya. J. Infus. Nurs. 2007 30:168-172). These conditions may be life threatening for patients. Chemotherapy is often compromised by the presence of or subsequent development of resistance, which can often span different classes of drugs (multidrug resistance).
Immunotherapy for cancer has been employed for many years. One of the first immune treatments was a mixed bacterial vaccine (Coley's vaccine), the active ingredient of which is bacterial lipopolysaccharide. It should be noted that regulatory authorities strive to limit or eliminate the presence of lipopolysaccharide from pharmaceutical agents due to unwanted and often toxic effects. More recently, mixtures of irradiated malignant melanoma cells have been used to induce immune responses in patients with malignant melanoma, which increased survival in several patients (Morton, et al. Ann. Surg. 1992, 216:463-482). One major benefit offered by immune therapy (immunotherapy) is that it is not generally associated with the side effects of surgery, radiation or chemotherapy. In three studies using dendritic cell immunotherapy in patients with cancer, minimal to no side effects were reported (Hsu, et al. Nature Medicine, 1996 2:52-58; Murphy, et al. The Prostate, 1996 29:371-380; Nestle, et al. Nature Medicine, 1998, 4(3):328-332).
Mycobacterial cell walls are known to stimulate host immune defense mechanisms (both innate through the interaction with pathogen associated molecular pattern receptors—PAMPs, and acquired through the presence of immunogenic molecular species). Immunotherapy using whole, viable mycobacteria is used clinically in the treatment of bladder cancer. The mycobacterium bacillus Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis, is repeatedly instilled into the bladder of individuals with bladder cancer, where possible and preferably in an adjuvant setting following tumor removal by surgery (as described in for example the European Association of Urology Guidelines 2007 edition, pages 8-9). Its use however is associated with a range of adverse side effects related to its viable nature (Koya, et al. J. Urology 2006, 175:2004-2010) as well as an often low clinical efficacy and duration of response rate, especially in patients who experience treatment relapse (Witjes and Hendricksen. Eur. Urol. 2008, 53:24-26). Its use for the treatment of other cancers is contra-indicated because it contains live mycobacteria, and can give rise to fatal systemic infections (Orifice, et al. Tumori 1978, 64:437-443). Immunotherapy of cancer using intact but inactivated mycobacteria has been attempted using the mycobacterium Mycobacterium vaccae, but no definitive long-term survival following its use has so far been identified in clinical studies (see Stanford et al., Eur. J. Cancer 2008, 44:224-227). It is clear that intact mycobacteria, whether viable or inactivated, do not represent the most effective form of immunotherapy for the treatment of cancer.
Immunotherapy utilizing bacterial cell walls and bacterial extracts has been extensively evaluated in animal tumor models, in patients suffering from cancer (U.S. Pat. Nos. 4,503,048, 5,759,554 and 6,326,357), and as treatments for infectious diseases, such as bacterial and viral infections (U.S. Pat. Nos. 3,172,815, and 4,744,984).
Mycobacterial cell wall compositions with immune stimulant and anticancer activity (for example as described in U.S. Pat. Nos. 4,503,048, 5,759,554 and 6,329,347 or in Ribi et al., J. Bacteriol. 1965, 91:975-983) suffer from the disadvantage that biological reagents and materials, chemical reagents, solvents or diluents and enzymatic treatments are required for their preparation, with the potential for noxious chemical and foreign protein contamination. Moreover, it has been reported that in order to obtain optimal anticancer activity with highly purified mycobacterial cell walls (essentially consisting of the cell wall skeleton following extensive chemical and enzymatic treatments) formulation as oil emulsions is required (Yarkoni and Rapp, Cancer Res., 1979 39:535-7). Oil emulsions containing mycobacterial cell walls are often physically unstable and are difficult to prepare reproducibly, and can be toxic to the recipient because of the well-known potential to induce hypersensitivity reactions. Mycobacterial cell walls containing biologically active complexed DNA that possess both immunotherapeutic and anticancer activity and that do not depend on the presence of oil have also been described (U.S. Pat. No. 6,326,357), but these again suffer from the disadvantage that chemical and enzymatic treatments are required for their preparation, with the potential for noxious chemical and foreign protein contamination. In addition, using such compositions it has not proven possible to preferentially optimize either the immunotherapeutic activity or the anticancer activity.
It is recognized by those of ordinary skill in the art that disruption of microorganisms can be achieved using small sample volumes and refrigerated pressure cells (such as the Sorvall pressure cell) at high pressures of between 40,000-45,000 pounds per square inch (PSI, equivalent to 276-317 mPa) (see Ribi et al., J. Bacterial., 1966, 91:975-983). Such processes are time consuming, inefficient, and of low volume, and are additionally hampered by the current unavailability of this type of equipment. More efficient processes that use high pressure homogenization have been described for the isolation of proteins (as inclusion bodies) from genetically engineered microorganisms (see Peternel and Komel: Isolation of biologically active nanomaterial [inclusion bodies] from bacterial cells. Microbial Cell Factories 2010 9:66). It is however recognized by those of skill in the art that Gram-positive organisms are resistant to such processes by virtue of their peptidoglycan content and structure (see Diels and Michaels: High pressure homogenization as a non-thermal technique for the inactivation of microorganisms. Crit. Rev. Microbiol., 2006; 32:201-216). The use of techniques to minimize the number of homogenization cycles is also taught by those skilled in the art (see Bailey et al., Improved homogenization of recombinant Escherichia coli following pretreatment with guanidinium chloride: Biotech. Prog., 1995; 11:533-539). The use of such procedures is in fact clearly designed to remove cell wall fragments, not preserve them. The presence of nucleic acids such as DNA using high pressure disruption techniques is additionally taught as a contaminating material to be removed, not preserved (see Rathore et al., Analysis for residual host cell proteins and DNA in process streams of a recombinant protein product expressed in Escherichia coli cells: J. Pharm. Biomed. Anal., 2003; 32:1199-1211). What is needed are new procedures for the preparation of new bacterial nucleic acid compositions that use working volumes that are scalable, range from several mL to multi-liter volumes, and result in the efficient production of new bacterial nucleic acid and cell wall compositions.
It is known that mycobacterial cell walls and components thereof can stimulate and activate macrophages, monocytes and dendritic cells to produce bioactive molecules that can initiate, accelerate, amplify and stimulate responsive cells of the immune system such that an immune stimulatory effect is achieved. These bioactive molecules include, but are not limited to, hematopoietic and myeloid growth factors, cytokines and chemokines.
Growth factors are proteins that bind to receptors on a cell surface, with the primary result of activating cellular proliferation and/or differentiation. Cytokines are a unique family of regulatory proteins. Secreted primarily from cells of the immune system such as but not limited to leukocytes and acting as intercellular mediators, cytokines stimulate the humoral and cellular immune response, as well as the activation of phagocytic cells. Cytokines that are secreted from lymphocytes are termed lymphokines, whereas those secreted by monocytes or macrophages are termed monokines Many of the lymphokines are also known as interleukins (IL), since they are not only secreted by leukocytes but also able to affect the cellular responses of leukocytes. Chemokines are a class of cytokines that have the ability to attract and activate leukocytes, especially in response to infections (a process termed chemotaxis). They can be divided into at least three structural branches: c (chemokines, c), cc (chemokines, cc), and cxc (chemokines, cxc), according to variations in a shared cysteine motif (Johrer et al. Exp. Opin. Biol. Ther. 2008 8:269-290.). The harmful effects of chemotherapeutic agents or radiation therapy on the production of the cells of the immune system that are responsible for producing these hematopoietic and myeloid growth factors, cytokines and chemokines results in increased susceptibility to opportunistic infections.
Cancer (of which there are over 100 diseases) is an aberrant net accumulation of atypical cells, which results from uncontrolled cell division, an insufficiency of or defective apoptosis, or a combination of the two. Mutations in apoptosis-related genes such as, but not limited to, Fas, TNFR1 and p53/p21 have each been implicated in the pathogenesis of cancers (Levine, A. Cell 88:323-331, 1997; Fisher, D. Cell 78:529-542, 1994). Aberrant apoptosis is important not only to the pathogenesis of cancers, but also to a cancer's likelihood of resistance to many anti-cancer therapies.
Resistance to apoptosis induction has emerged as an important category of multiple drug resistance (MDR), one that likely explains a significant proportion of treatment failures. MDR, the simultaneous resistance to structurally and functionally unrelated classes of chemotherapeutic agents, can be both inherent and acquired. That is, some cancers never respond to therapy, whereas other cancers, initially sensitive to therapy, subsequently develop drug resistance through the selection of resistant clones. As chemotherapeutic agents rely primarily on an induction of apoptosis in cancer cells for their therapeutic effect, drug resistance, which diminishes the effectiveness of chemotherapeutic agents, leads directly or indirectly to reduced apoptosis and is generally associated with poor clinical prognosis in a variety of cancers.
Prior art anti-cancer agents have proven to be less than adequate for clinical applications. Many of these agents are inefficient (Bischoff et al. Science 274:373-376, 1996) or toxic, have significant side effects (Lamm et al. Journal of Urology 153:1444-1450, 1995), result in the development of drug resistance or immune sensitization and hypersensitivity reactions, and are debilitating for the recipient.
Therefore, there is a need for novel therapeutic compositions that stimulate responsive cells of the immune system to produce cytokines, chemokines and hematopoietic and myeloid growth factors, inhibit proliferation of cancer cells and induce apoptosis in cancer cells. These therapeutic compositions should be useful as an anti-cancer agent in their own right as well as having an adjuvant activity with respect to other anti-cancer agents. This therapeutic composition should be useful in preventing or treating myelosuppression associated with cancer, surgery, radiation or chemotherapy. Moreover, such a therapeutic composition should be simple and relatively inexpensive to prepare, its activity should be reproducible among preparations, its activity should remain stable over time, and its effects on cancer cells should be achievable with dose regimens that are associated with minimal adverse reactivity and toxicity. In addition, there is a need for methods of manufacturing novel therapeutic compositions that are efficient and that do not result in the presence of enzymes or chemicals used in their preparation.
There is also a need for a novel therapeutic composition that treats, prevents, abates or ameliorates autoimmune disorders, inflammatory or infectious disease, myelosuppression or hematopoietic and myeloid abnormalities. The therapeutic composition should be useful as an adjuvant with other therapeutic agents. Moreover, such a therapeutic composition should be simple and relatively inexpensive to prepare, its activity should be reproducible among preparations, its activity should remain stable over time, and its effects should be achievable with dose regimens that are associated with minimal toxicity.